Abstract
BackgroundNovel and more efficient compounds are urgently required for medical treatment of cystic echinococcosis (CE). Germinative cell culture of Echinococcus granulosus could be used for anti-echinococcosis agent tests and other biological studies on CE. This study was performed to establish an in vitro cell culture model for E. granulosus germinative cells and to evaluate the lethal effect of Zataria multiflora essential oil (ZMEO) on the cultured cells.MethodsThe inner surface of germinal layers of CE cysts was scraped, and the obtained materials were trypsinized to obtain a suspension of single germinative cells. Medium 199 was used as the basic culture medium and was supplemented with fetal bovine serum, 2-mercaptoethanol, l-cysteine, l-glutamine, glucose, sodium pyruvate, hydatid fluid, amphotericin B and antibiotics. The cells were cultured at a concentration of 104 cells/ml of culture medium and incubated at 37 °C. The culture medium was replaced every 7 days. Chemical composition of ZMEO was identified by GC-MS analysis. ZMEO was tested at concentrations of 0.5–8 mg/ml. Viability of the cells was assessed by trypan blue exclusion assay.ResultsA significant increase in the cell number was evident at 20, 30 and 45 days after cultivation. At 45 days of cultivation, the number of cells was approximately five-fold higher than on the first day. In GC-MC analysis, carvacrol, p-cymene, g-terpinene and thymol were found to be the main compounds of ZMEO. The lethal effect of ZMEO on the germinative cells at concentrations of 6, 7 and 8 mg/ml was 100% after 60, 25 and 7 min of exposure, respectively.ConclusionsAt 45 days of cultivation, the cell concentration was suitable for the desired in vitro experiments. A high lethal effect of ZMEO on the germinative cells of E. granulosus may be considered an opportunity for the introduction of a novel, more effective and safer therapeutic agent for treatment of CE using an herbal product.Graphical
Highlights
Novel and more efficient compounds are urgently required for medical treatment of cystic echinococcosis (CE)
A gradual increase in the dividing cells was observed at 7 and 15 days after the start of cultivation (Fig. 1a, b), and a significant increase in the cell number was evident at 20, 30 and 45 days after incubation (Fig. 1c–e) so that at 45 days of cultivation the number of cells increased to 5 × 104 cells/ml culture medium
The wall of the larval form of E. granulosus (CE cyst) has two layers: an acellular external laminated layer and an internal germinal layer consisting of multiplying cells, which may differentiate into protoscoleces or daughter cysts
Summary
Novel and more efficient compounds are urgently required for medical treatment of cystic echinococcosis (CE). Germinative cell culture of Echinococcus granulosus could be used for anti-echinococcosis agent tests and other biological studies on CE. The disease may be treated by surgery, puncture, aspiration, injection, re-aspiration (PAIR), anti-parasitic treatment or watch and wait for inactive cysts [5, 6]. Albendazole, as the drug of choice for anti-parasitic treatment of CE [2], should be administered for long periods and at high doses [8]; it may be accompanied by adverse side effects in the patients [9]. Novel and more efficient compounds are urgently required for medical treatment of CE [11,12,13]
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